Characterization, in-silico, and in-vitro study of a new steroid derivative from Ophiocoma dentata as a potential treatment for COVID-19

The medicinal potential of marine invertebrates' bioactive components that may act as anti-COVID-19 demonstrated promising results. Ophiocoma dentata, which is common in the Red Sea, is one such source. Therefore, this study aimed to isolate a new compound from the brittle star, Ophiocoma dentata, and evaluate its efficacy as anti-COVID-19 in-silico and in-vitro. Standard procedures were followed in order to assess the isolated compound’s preliminary toxicity and anti-inflammatory properties. Computer virtual screening technology through molecular docking and ADMET studies was conducted as well as a new steroid derivative was isolated for the first time, named 5α-cholesta-4(27), 24-dien-3β, 23 β-diol. Investigation of the Anti-Covid-19 activity of the isolated compound using a Plaque reduction assay revealed 95% inhibition at a concentration of 5 ng/µl (12.48 µM). Moreover, this compound showed an IC50 of 11,350 ± 1500 ng/ml against the normal fibroblast cells, indicating its safety. Interestingly, this compound exhibited anti-inflammatory activity with an IC50 of 51.92 ± 0.03 μg/ml compared to a reference drug’s IC50 of 53.64 ± 0.01 μg/ml, indicating that this compound is a potent anti-inflammatory. In silico data have proved that the isolated compound is a promising viral inhibitor against SARS-CoV2 and is thus recommended as a future nature preventive and curative antiviral drug.

www.nature.com/scientificreports/ production of a highly abundant variety of biologically active substances 5 . Echinoderms demonstrate as an untapped source in an attempt to identify novel and beneficial products 6 . According to Fayed et al. 7 , marine natural products have demonstrated antiviral activity against coronaviruses, and various aquatic species have previously been discovered to produce a variety of antiviral lead compounds. Brittle stars (Ophiuroidea) are the largest echinoderm group in terms of species, as well as the most frequent 8 . There have been a few studies on the biological activity of brittle star species have been published. However, the antiviral activity of the brittle star revealed that the most abundant bioactive metabolites derived from brittle star are steroidal glycosides, carotenoid sulfates, and naphthoquinone. All of which are essential for life maintenance on earth and have the potential to be used in biomedicine 8 . Molecular modeling and virtual screening are examples of computational-based techniques that contribute to understanding the molecular components of protein-ligand interactions throughout the rational drug discovery process 9,10 . Two intriguing therapeutic targets for SARS-CoV-2 related disorders are the main protease (Mpro) and the RNA-dependent RNA polymerase (RdRP), which are responsible for the viral polyprotein proteolytic process along with viral genome replication and transcription 11,12 . These two target sites have been intensively docked to develop or distinguish structure-based effective medicines for COVID-19 based on their critical role in the life cycle of SARS-CoV-2 13 . Natural bioactive compounds are currently being screened for their affinity for COVID-19 molecular targets using molecular docking, with the advantage that natural products are free of harmful or adverse effects 14,15 . This study aims to isolate a new compound from the brittle stars, Ophiocoma dentata that can resist COVID-19. In addition, structure elucidation, anti-inflammatory activity, molecular docking and in silico ADMET studies were performed.

Results
Structure determination of the isolated compound. The new compound was obtained as yellow crystals, and HREIMS provided its molecular formula, C 27  The 13 C NMR spectrum of the isolated compound revealed the resonance of 27 carbons identified as four methyls, ten methylene (including two terminal double bonds), one oxygenated methine, and five quaternary carbons. The isolated compound's complete 1 H and 13 C NMR chemical shifts were assigned using a combination of COSY-45°, HSQC, and HMBC spectra. These spectroscopic studies suggested that the isolated compound had a steroidal type skeleton. Consequently, were proposed the structure for this new compound named 5α-cholesta-4(27), 24-dien-3β, 23 β-diol. 1 H NMR, 13 C NMR, HSQC, HMBC, COSY, NOESY, and FTIR data are depicted in (Fig. 1 and supplementary Figs. S2-S8; Table S1). Molecular docking, ADMET analysis, and in silico toxicity studies of the isolated compound. In this work, the binding potential of the isolated compound against three proteins of SARS-CoV-2 has been investigated. The selected proteins are: (i) COVID-19 main protease (M pro ) (PDB ID: 6lu7, resolution: 2.16 Å), (ii) nonstructural protein (nsp)10 (PDB ID: 6W4H, resolution: 1.80 Å), and (iii) RNA-dependent RNA polymerase (PDB ID: 7BV2, resolution: 2.50 Å).
Re-docking processes of the co-crystallized ligands (PRD_002214, SAM, and F86) were preceded against the active pockets of COVID-19 main protease, NSP10, and RNA-dependent RNA polymerase, respectively, to validate the docking procedure. The calculated RMSD values between the re-docked poses and the co-crystallized ones were 3.10, 1.07, and 1.34, indicating the efficiency and validity of the docking processes (Fig. 2).
Determination of anti-SARS-CoV2, MTT cytotoxicity, and anti-inflammatory activity. The percent inhibition of various concentrations of the isolated compound against SARS-CoV2 is shown in (Table 1).  In this study, human gingival fibroblast cell lines (Fig. 3) were exposed to different concentrations of the isolated compound. The cellular responses were determined using in vitro toxicity MTT assay. The changes in the morphology of cells exposed to different concentrations of the isolated compound were monitored using inverted microscopy. The results demonstrated that the cells exposed to 12.5 μg/ml of the isolated compound gradually lost their characteristic phenotype, started to shrink, and obtained irregular shapes. Percentage inhibition of the normal fibroblast cell growth at different concentrations of the isolated compound is shown in (supplementary Fig. S9), with an IC 50 value of 11.35 ± 1.5 μg/ml (0.02 mM).

Discussion
Chemical studies on Ophiocoma dentata have resulted in discovering a new steroid, 5α-cholesta-4(27), 24-dien-3β, 23 β-diol. The structure of the isolated compound was determined with the help of spectroscopic studies. The isolated compound's NMR spectra matched those of 5 α-cholesta-9(11),24-dien-3 β,6 α,20β-triol-23-one 3-sulphate, reported by Yang et al. 16 , except for an absence of hydroxyl group at C-6, C-20 position and the double bond between C-9 and C-11 as well as additional double bond at C-4 and this was confirmed by the HMBC experiment. Inspection of its NMR data indicated a considerable similarity to those of ophidianoside F 17 except for an absent hydroxyl group at C-6, C-20 and the double bond between C-9 and C-11, and the presence of a double bond between C-24 and C-25 position.
Docking studies were carried out using MOE14.0 software, yielding free energy (ΔG) values that indicate the examined molecule's binding interaction with the selected protein.
The docking mode of the co-crystallized ligand (PRD_002214) and COVID-19 main protease formed four hydrogen bonds and three hydrophobic interactions. The first pocket of M pro was occupied by 2-oxopyrrolidin-3-yl moiety, forming two hydrogen bonds with Thr190 and Gln189. Additionally, the isopropyl moiety occupied the second pocket of M pro forming three hydrophobic interactions with His41 and Met165. Furthermore, the third pocket was occupied by the benzyl acetate moiety, whereas the 5-methylisoxazole-3-carboxamide moiety was buried in the fourth pocket, forming a hydrogen bond with Thr26. Finally, one hydrogen bond was formed between an amide group and Met165 (supplementary Fig. S10).
Considering the isolated compound's binding mode against the COVID-19 main protease protein, it occupied three pockets of the protein with a similar orientation to that of the co-crystallized ligand inside the active pocket of M pro . Exhaustively, the (S)-2-methylenecyclohexan-1-ol moiety occupied the first pocket of M pro , forming one hydrophobic interaction with Met49. Moreover, the (3aR,7aS)-3a-methyloctahydro-1H-indene moiety was buried in the second pocket, forming a hydrogen bond with His41. In addition, the (R)-3-methylhex-1-en-3-ol moiety was incorporated in hydrophobic interaction with Met165 in the third pocket of M pro (Fig. 4).
The compound's interaction with the active pocket of NSP10 formed three hydrophobic interactions and a hydrogen bond. Closely, the hydroxyl group of (S)-2-methylenecyclohexan-1-ol moiety formed a hydrogen bond Asp6912. Such moiety formed two hydrophobic interactions with Leu6898. In addition, the (3aR,7aS)-3a-methyloctahydro-1H-indene moiety formed one hydrophobic interaction with Pro6932. The orientation of the compound inside the active pocket is similar to that of the co-crystallized ligand to some extent (Fig. 5).
ADMET studies were carried out for the isolated compound using lopinavir as a reference compound. Discovery studio 4.0 was used to predict the following ADMET descriptors; blood-brain barrier (BBB) penetration, aqueous solubility, intestinal absorption, hepatotoxicity, cytochrome P450 inhibition, and plasma protein binding. The predicted descriptors are listed in (Table 4). www.nature.com/scientificreports/ ADMET -BBB penetration studies predicted that the isolated compound possesses a high level compared to lopinavir, which is very low level. Although the compound showed a very low level of ADMET aqueous solubility, it was predicted to have a good level of intestinal absorption. The isolated compound was predicted to be a non-hepatotoxic and non-inhibitor of CYP2D6. Consequently, the liver dysfunction side effect is not expected upon administration of this compound. The plasma protein binding model predicts that the isolated compound can bind plasma protein over 90% (Fig. 7).
The isolated compound's toxicity was predicted using the validated and constructed models in the Discovery studio 4.0 software 18,19 as follows: (i) FDA rodent carcinogenicity which expects the probability of a molecule to be a carcinogen. (ii) The tumorigenic dose rate 50 (TD50) of a chemical in a rodent chronic exposure toxicity test is predicted by the carcinogenic potency TD50 20 . (iii) The rat maximum tolerated dose (MTD) of a chemical is estimated using Rat maximum tolerated dose 21,22 . (iv) In the toxicity test of a chemical, the rat oral LD50 predicts the rat oral acute median lethal dose (LD50) 23 . (v) The rat chronic LOAEL predicts a compound's rat chronic lowest observed adverse effect level (LOAEL) value 24,25 . (vi) Ocular irritancy predicts if a certain compound is probable to be an ocular irritant and how severe the irritation will be in the Draize test 26 . (vii) skin irritancy predicts if a compound would irritate the skin and how severe it will be in a rabbit skin irritancy test 26 .
The isolated compound exhibited in silico low adverse effects and toxicity towards the tested models as shown in (Table 5). About FDA rodent carcinogenicity, the tested compound was predicted to be non-carcinogenic. For carcinogenic potency TD 50 rat model, the tested compound showed TD 50 value of 0.621 mg/kg body weight/day which is less than lopinavir (TD 50 = 3.553 mg/kg body weight/day). Regarding the rat maximum tolerated dose model, the tested compound showed a maximum tolerated dose value of 0.040 g/kg body weight which is less than lopinavir (0.117 g/kg body weight). The examined compound demonstrated an oral LD 50 value of 1.715 mg/ kg body weight/day which is higher than lopinavir (1.154 mg/kg body weight/day). For the rat chronic LOAEL model, the isolated compound showed a LOAEL value of 0.002 g/kg body weight which is less than lopinavir (0.049 g/kg body weight). Moreover, the compound was predicted to be an irritant in the ocular irritancy model. Finally, it was expected to have moderate irritancy against the skin irritancy model.
According to Mostafa et al. 27 , Azithromycin, which is an FDA-approved antimicrobial drug with promising antiviral activity for repurposing against COVID-19, its mode of action was during the viral replication, which demonstrated up to 70% inhibition at concentration 10.4 µM and exhibited moderate virucidal effect with 37% viral inhibitory effect. Additionally, the Niclosamide drug exhibited a high virucidal effect with a 78% viral www.nature.com/scientificreports/ inhibitory effect at concentration 10.4 µM, suggesting that our isolated compound is a promising viral inhibitor against SARS-CoV2 and its mode of action could be during the viral replication or had a virucidal effect. The results revealed that the isolated compound has low toxicity to normal cells and could be used as an antiviral agent after performing in vivo assays. Interestingly, the effective dose which inhibited 95% of the viral count was 5 ng/µl (5 μg/ml) and that below the IC 50 value (11.35 ± 1.5 μg/ml) of the normal fibroblast cells. The obtained IC 50 for the isolated compound was lowered than the reference drug (Diclofenac), indicating that the compound was more potent than diclofenac and could be used as an anti-inflammatory drug.
Echinoderms are rich bioactive components that provide tremendous pharmaceutical and clinical medicine values. Similarly, like many marine invertebrates, survival requirements have led to the evolution of these complex substances. Interestingly, several secondary metabolites were proven to have a potent antiviral activity as well as a relatively non-cytotoxicity. As a result, these promising echinoderm natural compounds could be used to  Table 4. The predicted ADMET for the isolated compound and lopinavir. a 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low. b 1 = very low, 2 = low, 3 = good, 4 = optimal. c 0 = good, 1 = moderate, 2 = poor, 3 = very poor. d TRUE = inhibitor, FALSE = non inhibitor. e FALSE means less than 90%, TRUE means more than 90%. www.nature.com/scientificreports/ develop new antiviral drugs. In this study, 5α-cholesta-4(27), 24-dien-3β, 23 β-diol was isolated from the brittle stars, Ophiocoma dentata showing 95% inhibition of COVID-19 virus at concentration 5 ng/µl, which is safe to the normal cells. The current study's findings are promising regarding finding an effective cure for the COVID-19 pandemic. Further in-vivo studies regarding the clinical and structure-activity relationship are needed to confirm the isolated compound's potential against the infectious virus SARS-CoV-2.

BBB level a Solubility level b Absorption level c CYP2D6 prediction d Hepatotoxicity PPB prediction e
Marine natural products remain a promising source for discovering high structural diversity and various bioactivities that can be directly developed or used as starting points for the development of novel medications. In the future, we must consider the final dosage form for human use (e.g., tablets, capsules, and injections) to have the potential to be translated into a clinically applicable therapeutic. Biological challenges, large-scale manufacturing, biocompatibility, intellectual property, stable storage, government regulations, and overall costeffectiveness should all be considered during clinical development. Future research could focus on determining the biodistribution, Pharmacodynamics, and pharmacokinetics in humans, as well as screening for safety and demonstrating the preliminary efficacy of the isolated compound. It is critical to comprehend how the in-vivo environment influences its long-term structural organization, retention, and clearance.   Spectroscopic characterization of the isolated compound. NMR analysis. The 1 H NMR and 13 C NMR analysis was measured at Bruker AVANCE 400 MHZ NMR spectrometers at the University of Winnipeg, Canada, using Deuterated chloroform (CDCl 3 ). COSY, HSQC, HMBC, and NOESY were also measured. Chemical shift (δ) values were given in ppm while coupling constants (J) in Hz.

Methods
FT-IR analysis. The compound's functional groups were identified using infrared spectroscopy. The powdered compound was mixed 1:1 with dried KBr powder to make a compressed pellet using the Bruker Alpha Fourier transform infrared spectroscopy spectrometer with the subsequent recording of infrared spectrum in the transmission mode from 4000 to 400 cm −1 .
GC-MS analysis. The compound was subjected to GC-MS to identify and confirm the structure of the purified bioactive compound. The sample was analyzed in an Agilent 7890A-5975C high-resolution gas chromatography-mass spectroscopy instrument. The carrier gas was Helium, and the analysis was performed at 90-300 °C. The identification and confirmation of the structure of the compound were made using computer matching of spectral data with that of standards.  29 . The free energies and binding modes of the tested molecules against target proteins were established in this investigation. Water molecules were first eliminated from target proteins crystal structures, leaving only one chain required for binding. The reference ligands were co-crystallized. The hydrogen atoms were then hidden, and the protein structures were protonated. Afterward, the MMFF94x force field was used to reduce the energy. After that, the binding pockets of each protein were defined [30][31][32] .
ChemBioDraw Ultra 14.0 was used to draw the structure of the investigated compound and the co-crystallized ligands, which were saved as SDF formats. Subsequently, the stored files were opened using MOE software, and 3D structures were protonated. The MMFF94x force field was then applied to minimize the energy of the molecules. Validation processes for each target receptor were carried out by running the docking process for only the co-crystallized ligand. Valid performance is indicated by low RMSD values between docked and crystal conformations 33,34 . The output from the MOE software was further analyzed and visualized using the Discovery Studio 4.0 software 35-38 . In silico ADMET analysis. Discovery studio 4.0 was used to determine the compound's ADMET descriptors (absorption, distribution, metabolism, excretion, and toxicity). According to the small molecule preparation protocol, the compound was prepared and minimized after applying the CHARMM force field. These studies were then carried out using the ADMET descriptors procedure 33,36 . In silico toxicity studies. Discovery studio 4.0 was used to calculate the toxicity parameters of the isolated compound. Lopinavir was used as a reference drug. The toxicity prediction (extensible) protocol calculated various parameters 36,39,40 . Determination of anti-SARS-CoV2 activity. In order to evaluate the antiviral activity of the isolated compound, the plaque reduction assay was carried out according to Mustafa et al. 27 at the main laboratories of chemical warfare in Egypt. Percentage reduction in plaques formation in comparison to control wells was recorded as follows: MTT cytotoxicity assay. MTT assay, an in-vitro cytotoxicity test, was used to measure cell viability and membrane damage following van de Loosdrecht et al. 41 . Sample dissolved in 10% DMSO, then tested against human gingival fibroblast cell lines (obtained as a gift from faculty of medicine, Egypt) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). A microplate reader (ASYS-EXPERT 96) was utilized to measure the optical density at 540 nm. The changes in the morphology of cells exposed to the isolated compound were monitored using inverted microscopy and Biorad (ZOE fluorescent cell imager). www.nature.com/scientificreports/ Anti-inflammatory activity. The isolated compound's anti-inflammatory activity in-vitro was assessed by the human red blood corpuscles (HRBCs) membrane-stabilizing method 42 . The percentage membrane stabilization was calculated using the following formula: Statistical analysis. SPSS version 16 was used to analyze the data obtained using one-way analysis of variance (ANOVA). Values are expressed as mean ± SD (n = 3). P values ≤ 0.05 were considered significant.

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary  Information files).